BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-beta family of growth factors. Disruption of BMP2 signaling results in developmental defects, cancers, and other diseases. BMP2 mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct 3'-untranslated regions. The longer mRNA contains additional putative binding sites for post-transcriptional regulatory factors, including micro-RNAs. We combined functional assays with computational analyses of emerging genome data to define site- and species-specific polyadenylation determinants. In all mouse and human cell lines tested, shorter mRNAs resulting from using the first polyadenylation signal (PA1) were more abundant than mRNAs from the second signal (PA2). However, the PA1/PA2 usage ratios were 2-3-fold higher in human than in mouse cells. Expression of human BMP2 constructs in mouse cells and mouse constructs in human cells showed that cis-regulatory elements direct species-specific 3' processing of BMP2 transcripts. A 72-nucleotide region downstream of PA2 in the mouse sequence contains two novel cis-acting elements previously hypothesized to regulate polyadenylation in a bioinformatics analysis. Mutations that humanized the mouse-specific elements lowered the affinity for cleavage stimulation factor CstF64 and significantly weakened the PA2 signal relative to the PA1 signal. Thus, we have experimentally defined for the first time cis-regulatory elements that control a species-specific difference in the 3'-end processing of BMP2 and potentially of other genes.